Title

Study of the S. cerevisiae mitochondrial chaperonin, Hsp60, and the identification and characterization of SCS1/RTS1, a high-copy suppressor of Hsp60(ts)

Date of Award

1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Richard L. Hallberg

Keywords

SCS1/RTS1, molecular chaperone, Saccharomyces cerevisiae, Molecular biology, Cellular biology, Genetics

Subject Categories

Molecular Genetics

Abstract

In order to study the functions of the S. cerevisiae mitochondrial chaperonin, Hsp60, I have generated 25 temperature-sensitive lethal mutant alleles of the HSP60 gene. Mutant strains expressing these alleles manifested a wide variety of abnormal phenotypes, both at permissive and non-permissive temperatures.

To identify proteins functionally related to Hsp60, I identified genes which, when over-expressed, suppressed the temperature sensitive phenotype of cells expressing a HSP60 mutant allele. One particular gene, RTS1, was characterized in detail. RTS1 is not an essential gene, but RTS1-null strains are temperature sensitive. RTS1 encodes an 86 kDa cytoplasmic protein which is heavily modified in vivo. Over-expression of RTS1 had significant effects on the cellular levels of mRNAs encoding the heat-inducible (heat shock) proteins Cpn10 and Mge1p, two other mitochondrial protein co-chaperones, but not on mRNAs encoding a number of other mitochondrial or cytosolic heat shock proteins analyzed. It was this property, presumably, that enabled overexpression of RTS1 to suppress mutant alleles of HSP60.

Besides defining the role of RTS1 in regulating the expression of mitochondrial chaperones, I also identified its role in cell cycle control. First, CLB2, a gene encoding a regulatory subunit of protein kinase, was identified as a high-copy suppressor of the RTS1-null strain. The activity of Clb2 is required for controlling cell progression from G2 to M phase. Second, RTS1-null cells were observed to have a typical cell division cycle (cdc) mutant phenotype at high temperatures. Finally the RTS1 protein was found modified in a cell cycle dependent manner, implying that the specific activity of RTS1 is cell cycle dependent.

Human and rabbit homologs of RTS1 were identified by others and shown to encode regulatory (B) subunits of protein phosphatase 2A. As another S. cerevisiae B regulatory subunit gene, CDC55, had been previously described and as PP2A is essential for cell viability, I created a strain null for both genes. This strain was viable, suggesting there are more B subunit genes remaining to be identified. Analyses of this strain also showed that RTS1 and CDC55 play distinctly different roles in vivo.

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